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The formation of protein disulfide bonds in the Escherichia coli periplasm by the enzyme DsbA is an inaccurate process. Many eukaryotic proteins with nonconsecutive disulfide bonds expressed in E. coli require an additional protein for proper folding, the disulfide bond isomerase DsbC. Here we report studies on a native E. coli periplasmic acid phosphatase, phytase (AppA), which contains three consecutive and one nonconsecutive disulfide bonds. We show that AppA requires DsbC for its folding. However, the activity of an AppA mutant lacking its nonconsecutive disulfide bond is DsbC-independent. An AppA homolog, Agp, a periplasmic acid phosphatase with similar structure, lacks the nonconsecutive disulfide bond but has the three consecutive disulfide bonds found in AppA. The consecutively disulfide-bonded Agp is not dependent on DsbC but is rendered dependent by engineering into it the conserved nonconsecutive disulfide bond of AppA. Taken together, these results provide support for the proposal that proteins with nonconsecutive disulfide bonds require DsbC for full activity and that disulfide bonds are formed predominantly during translocation across the cytoplasmic membrane. The formation of protein disulfide bonds in the Escherichia coli periplasm by the enzyme DsbA is an inaccurate process. Many eukaryotic proteins with nonconsecutive disulfide bonds expressed in E. coli require an additional protein for proper folding, the disulfide bond isomerase DsbC. Here we report studies on a native E. coli periplasmic acid phosphatase, phytase (AppA), which contains three consecutive and one nonconsecutive disulfide bonds. We show that AppA requires DsbC for its folding. However, the activity of an AppA mutant lacking its nonconsecutive disulfide bond is DsbC-independent. An AppA homolog, Agp, a periplasmic acid phosphatase with similar structure, lacks the nonconsecutive disulfide bond but has the three consecutive disulfide bonds found in AppA. The consecutively disulfide-bonded Agp is not dependent on DsbC but is rendered dependent by engineering into it the conserved nonconsecutive disulfide bond of AppA. Taken together, these results provide support for the proposal that proteins with nonconsecutive disulfide bonds require DsbC for full activity and that disulfide bonds are formed predominantly during translocation across the cytoplasmic membrane. Disulfide bonds between cysteine residues make an important contribution to the folding pathway and stability of many proteins. Early in vitro studies on protein folding showed that the protein RNase when denatured and its disulfide bonds reduced could refold into an active enzyme with the correct disulfide bonds formed (1Anfinsen C.B. Haber E. Sela M. White Jr., F.H. Proc. Natl. Acad. Sci. U. S. A. 1961; 47: 1309-1314Crossref PubMed Scopus (897) Google Scholar). Although these results indicated that the information necessary for the proper folding of proteins was intrinsic to the amino acid sequence, the rate of disulfide bond formation in these experiments was much slower than the rates observed in living cells, and the yield of active enzyme was low. The low yields of properly folded enzyme were attributed to the formation of incorrect disulfide bonds in many of the assembled protein molecules. Thus, the in vitro studies, although remarkably successful, were unable to come close to replicating the rapid kinetics and the accuracy of in vivo disulfide bond formation. To explain this inaccuracy observed in biochemical experiments, Anfinsen and co-workers (1Anfinsen C.B. Haber E. Sela M. White Jr., F.H. Proc. Natl. Acad. Sci. U. S. A. 1961; 47: 1309-1314Crossref PubMed Scopus (897) Google Scholar) posited the existence in cells of an enzyme, protein-disulfide isomerase (PDI), 1The abbreviations used are: PDI, protein disulfide isomerase; ER, endoplasmic reticulum; ORF, open reading frame. which would increase the yields of active enzyme by correcting incorrectly formed disulfide bonds. Such an enzymatic activity was discovered in beef liver cells (2De Lorenzo F. Goldberger R.F. Steers Jr., E. Givol D. Anfinsen B. J. Biol. Chem. 1966; 241: 1562-1567Abstract Full Text PDF PubMed Google Scholar). The explanation for the slow in vitro rates was resolved much later with the discovery that in vivo an enzyme is required to catalyze the efficient formation of protein disulfide bonds (3Bardwell J.C. McGovern K. Beckwith J. Cell. 1991; 67: 581-589Abstract Full Text PDF PubMed Scopus (830) Google Scholar, 4Kamitani S. Akiyama Y. Ito K. EMBO J. 1992; 11: 57-62Crossref PubMed Scopus (225) Google Scholar, 5Peek J.A. Taylor R.K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6210-6214Crossref PubMed Scopus (184) Google Scholar). Although protein-disulfide isomerase (PDI) was originally detected in the endoplasmic reticulum (ER) of eukaryotes as an enzyme that catalyzes disulfide bond isomerization in vitro, its in vivo role is still unclear. PDI is required for the formation of disulfide bonds in the endoplasmic reticulum (ER) (6Frand A.R. Kaiser C.A. Mol. Cell. 1998; 1: 161-170Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 7Pollard M.G. Travers K.J. Weissman J.S. Mol. Cell. 1998; 1: 171-182Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). However, it is not known whether PDI carries out both oxidation and isomerization reactions in the ER or whether the latter reaction might be performed by other ER proteins. In Gram-negative bacteria, the enzyme catalyst of disulfide bond formation is the protein, DsbA, which is located in the periplasmic space of bacteria (3Bardwell J.C. McGovern K. Beckwith J. Cell. 1991; 67: 581-589Abstract Full Text PDF PubMed Scopus (830) Google Scholar, 4Kamitani S. Akiyama Y. Ito K. EMBO J. 1992; 11: 57-62Crossref PubMed Scopus (225) Google Scholar, 5Peek J.A. Taylor R.K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6210-6214Crossref PubMed Scopus (184) Google Scholar). A separate protein, DsbC, carries out protein disulfide bond isomerization (8Shevchik V.E. Condemine G. Robert-Baudouy J. EMBO J. 1994; 13: 2007-2012Crossref PubMed Scopus (132) Google Scholar, 9Missiakas D. Georgopoulos C. Raina S. EMBO J. 1994; 13: 2013-2020Crossref PubMed Scopus (193) Google Scholar). DsbC is a “Y”-shaped homodimer with two disulfide bonds in each 20-kDa monomer (10McCarthy A.A. Haebel P.W. Torronen A. Rybin V. Baker E.N. Metcalf P. Nat. Struct. Biol. 2000; 7: 196-199Crossref PubMed Scopus (219) Google Scholar). The C-terminal disulfide bond (Cys140-Cys163) is required for folding and stability of DsbC, whereas the N-terminal redox active cysteines (Cys98-Cys101) are required for the isomerase activity and are maintained in a reduced form by the inner membrane protein DsbD (11Rietsch A. Bessette P. Georgiou G. Beckwith J. J. Bacteriol. 1997; 179: 6602-6608Crossref PubMed Scopus (194) Google Scholar). It appears that cysteine 98 attacks incorrect disulfide bonds in misfolded substrates, and the resultant mixed disulfide between enzyme and substrate is resolved by cysteine 101 (11Rietsch A. Bessette P. Georgiou G. Beckwith J. J. Bacteriol. 1997; 179: 6602-6608Crossref PubMed Scopus (194) Google Scholar). The pairs of N-terminal cysteines (Cys98-Cys101) of the dimeric DsbC face each other in a hydrophobic cleft formed by the two arms of the Y shape. Because misoxidized substrates of DsbC are likely to be misfolded with core hydrophobic residues surface-exposed, the hydrophobic cleft of DsbC might mediate the interaction between the misfolded substrate protein and cysteines 98-101. Furthermore, DsbC possesses chaperone activity that is independent of cysteines 98 and 101 (12Chen J. Song J.L. Zhang S. Wang Y. Cui D.F. Wang C.C. J. Biol. Chem. 1999; 274: 19601-19605Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Joly and Swartz (38Joly J.C. Swartz J.R. Biochemistry. 1997; 36: 10067-10072Crossref PubMed Scopus (118) Google Scholar) reported that dependence on DsbC for proper folding an eukaryotic proteins expressed in Escherichia The proteins that not require DsbC disulfide bonds that were formed between cysteines found in consecutive in the proteins. In the proteins nonconsecutive disulfide the latter of proteins a disulfide bond between two cysteines that between in the amino acid cysteine or cysteines that is in a disulfide bond the disulfide bond between cysteines and in the substrates used to the isomerase activity of DsbC eukaryotic in The that eukaryotic proteins expressed in E. coli were in studies on DsbC is that of the known proteins of the bacteria disulfide bonds. and A. J.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) the E. coli periplasmic proteins RNase and as proteins stability is or in lacking DsbC. In this we a to as a substrate of DsbC the E. coli periplasmic protein phytase protein was it contains disulfide one of which a of cysteines located in the amino acid mutant of AppA and engineering of an AppA homolog, Agp, we the proposal that disulfide bond a role in a protein We the of a for the of disulfide bond formation during protein and and used in this are in and were and J. A Scholar). were in A. D. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) with the of AppA and Agp were with and used in this or or or A. Bessette P. Georgiou G. Beckwith J. J. Bacteriol. 1997; 179: 6602-6608Crossref PubMed Scopus (194) Google G. S. K. E. PubMed Scopus Google D. Beckwith J. J. Bacteriol. PubMed Scopus Google into with a G. S. K. E. PubMed Scopus Google into to in to in to in to in to in to in to in to in and to in and to in and to in and to in into of in with of in with of in with in a To a or a we the D. Beckwith J. J. Bacteriol. PubMed Scopus Google Scholar). 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E. coli were in of of of of and of in of with and in an Because of the in AppA is found predominantly in the were by for The was to of to The proteins in the were by to a of and to for The was in and and in of the for The was three The in the formation of protein that were by for The protein was to a and the protein was into with an were for activity and in an that showed activity and were and used to in for of acid phosphatase activity for AppA and Agp were with the substrate as E. E. Mol. PubMed Scopus Google Scholar) with were performed in for Agp J. PubMed Scopus Google Scholar) or for AppA C. Beckwith J. J. Bacteriol. PubMed Google Scholar) of cells or were in the of in of cells were with of and the reaction was with the of The acid phosphatase activity was by the of the formed was as in with activity in Scholar). experiments were in and the in vivo redox of the were with redox acid indicated the proteins were reduced with the A. D. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of in E. with in were The were to as and and are the but in other information in the has The as are the proteins are with but to one of the for of on studies that the of disulfide bonds in a protein was a in whether it would be a substrate for DsbC, we performed a for proteins known to nonconsecutive disulfide bonds in the native for DsbC, E. the we for E. coli periplasmic proteins nonconsecutive disulfide bonds on a protein, this AppA was as a acid phosphatase C. Beckwith J. J. Bacteriol. PubMed Google Scholar) that E. M. B. J. Biol. Chem. Full Text PDF PubMed Google Scholar). It its activity and with acid as a substrate and has as a phytase S. Wang G. Zhang J. J. 2000; PubMed Scopus (132) Google Scholar). D. S. Nat. Struct. Biol. 2000; 7: PubMed Scopus Google Scholar) the of disulfide one of nonconsecutive The RNase by and A. J.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) contains nonconsecutive disulfide bonds but was not is still in the of E. coli for with nonconsecutive the and the the the on an is than the of the the bonds the are by of the and the and the the the on an is than the of the the bonds the are by of the and in a AppA a of the in vivo DsbC dependence of we the into the it is expressed an was into and mutant The in which we these studies, contains the that is not expressed cells for the a in AppA acid phosphatase dependence of AppA on its disulfide bonds for folding into an active for the a in AppA activity that of its cysteines are misoxidized by DsbA, the isomerization activity of DsbC. However, we to out the that the in AppA in the was of the chaperone activity of the protein than the isomerase To this we expressed a mutant of DsbC with its active cysteines to and we whether this protein, its isomerase but not its chaperone activity (11Rietsch A. Bessette P. Georgiou G. Beckwith J. J. Bacteriol. 1997; 179: 6602-6608Crossref PubMed Scopus (194) Google was to full AppA The AppA activity in the this mutant a low that the in the was of the disulfide isomerase activity not were out on the to the and redox of AppA of AppA protein were observed in cells and cells of of or that the reduced protein or misoxidized protein is not by of by acid showed that of the cysteines in AppA are reduced in the of the periplasmic In cells of the isomerase DsbC, the redox of AppA was similar to that of results that correct disulfide bond formation in AppA is for its folding into an active than its We the experiments with of AppA a to a with a and we observed the dependence on DsbA and DsbC not results were when the experiments were in a not that the of the is not expressed in U. PubMed Scopus Google Scholar). We whether a periplasmic disulfide bond F. U. J.C. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google might a role in isomerization of AppA. However, of the on AppA activity not Furthermore, a of both and showed of activity than the mutant that the activity of AppA in the mutant was not to activity not of AppA that the of DsbC in activity of AppA that of the disulfide bonds of the enzyme are required for its proper or that a misoxidized disulfide bond with that To to which disulfide bonds are for AppA we in which each of the cysteine residues was to of the mutant AppA proteins reduced activity with of the cysteine than activity in activity could be to one of two to disulfide bond formation. The cysteines are of a disulfide bond that is important for the proper folding of AppA. The the of its in a disulfide form incorrect disulfide bonds with other cysteines in the To the role of the disulfide bonds of AppA in its we mutant of AppA each of the cysteine pairs in disulfide bonds was to with the cysteine of the mutant reduced of AppA Although and of the cysteine mutant of AppA activity of the cysteine of AppA than the The in between the and be to the of cysteines in the that could the of incorrect disulfide as The misoxidized with the cysteine are likely misfolded and to as indicated by the is not the when both the cysteines are cysteines and which are in a disulfide showed the of activity for and for in to was not as a mutant of AppA has in a for stability studies of AppA E. 2000; PubMed Scopus Google Scholar, E. J. PubMed Scopus Google Scholar). Disulfide of the of each lacking one of the disulfide bonds of but still of we an to the role of each disulfide bond on DsbC We the activity of AppA expressed in and mutant cells The results show that the mutant AppA lacking its nonconsecutive disulfide bond not require DsbC for its In lacking the or disulfide bonds dependence on DsbC. The AppA cysteine which the AppA activity of of the cysteine appears to show dependence on DsbC However, the low activity of this protein it to the of this it could be that the likely incorrect disulfide bond formed with the cysteine for is one of the cysteines of and that this the of in disulfide bond formation. are with the proposal that the nonconsecutive disulfide bond of AppA is the for the dependence of AppA on DsbC. It is that the amino acid of cysteines to in DsbC of AppA. To this we both cysteines and to The and proteins were independent of DsbC for activity not of the were by with The of AppA protein expressed by the mutant the disulfide bonds were between and of that with the AppA not of disulfide bonds in the protein to misfolded protein but not to a protein that is to The AppA Agp DsbC for into existence of a close of AppA with a of disulfide bonds has with a to the of the DsbC dependence of AppA. Agp is as a acid phosphatase and a of the phosphatase activity an of J. PubMed Scopus Google Scholar). Agp is to the periplasm and and to AppA The of Agp has J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google three consecutive disulfide bonds to of AppA but lacking the nonconsecutive disulfide bond in AppA The of the two AppA and Agp, are In this we used the of Agp to to its activity when expressed in lacking DsbC. The activity of Agp was dependent on DsbA, that Agp requires its disulfide bonds for activity However, it showed dependence on DsbC. were the was results are with the in which the dependence of AppA on DsbC for full activity is of its nonconsecutive disulfide it could be that the in the dependence of the two proteins on DsbC is to the between three and disulfide of consecutive However, and other proteins that are three disulfide the dependence of AppA be to the of disulfide bonds. the between AppA and Agp could be to a of both the and the of the cysteines in disulfide bonds. The of Agp to a a Disulfide on It DsbC results show that AppA is dependent on DsbC for its proper folding and that this dependence with the of the nonconsecutive disulfide We to whether the of a nonconsecutive disulfide bond into Agp would make Agp dependent on DsbC. of the AppA amino acid with that of Agp that are in the Agp in the the nonconsecutive disulfide bond in AppA amino and are conserved the Furthermore, of the of AppA and Agp that the are similar the disulfide on the and amino acid we in Agp with and in Agp with The or and into Agp were for dependence on DsbC. and the mutant Agp were expressed an in in or The of were and to Agp, both and activity when expressed in is of the that an of phosphatase, in which one of its cysteines has and a cysteine into the protein, dependent on DsbC M. Akiyama Y. Ito K. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). It is that a cysteine results in an that has a in the of DsbA, to form disulfide bonds with other disulfide bond could and of However, the that the which is the C-terminal of the DsbC dependence is with a formation of disulfide bonds the is on was observed to in activity to of that in a when expressed in a We that the proposal that a protein-disulfide bond isomerase is required for the proper folding of proteins that one or nonconsecutive disulfide bonds. proposal that the of DsbA or on substrate proteins to the of incorrect disulfide bonds when the protein to be assembled contains disulfide one of which is formed between cysteines located in the The results in with are to that as DsbA on substrate proteins to form disulfide bonds in a the one to the The in which this could is DsbA with cysteines in substrate proteins as are across the cytoplasmic membrane into the periplasmic Thus, DsbA would form disulfide bonds between cysteines that are located consecutively in the amino acid as it appears in the a nonconsecutive disulfide bond is required for the properly folded form of the protein, this of disulfide bond formation would to an incorrect disulfide the for disulfide bond which are found in the explanation for the formation and isomerization of disulfide bonds is on the Disulfide bond formation during protein Although this has not in bacteria, it is the in eukaryotic cells disulfide bond formation during translocation of proteins into the ER J. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, M. A. 1999; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Struct. 11: PubMed Scopus Google Scholar). DsbA is in its reactions with cysteines of substrate proteins. the that when proteins not into the periplasm are to disulfide bonds form that are not in the proteins in native (3Bardwell J.C. McGovern K. Beckwith J. Cell. 1991; 67: 581-589Abstract Full Text PDF PubMed Scopus (830) Google Scholar, J. Bacteriol. PubMed Google Scholar). that of in the periplasm not and that these are the on which DsbA is DsbA this the of the folded of the protein would as to which cysteines into disulfide bonds. the of nonconsecutive disulfide bonds is as the E. coli periplasmic protein, phytase (AppA), contains three consecutive disulfide bonds and one nonconsecutive We found that this protein a in vivo dependence on DsbC for its full of in cells for a in Furthermore, we show of active cysteine of DsbC that this dependence requires the disulfide bond isomerase activity of DsbC. substrates of DsbC eukaryotic proteins into the bacteria, this the for the of a native substrate for the isomerase activity of DsbC. We it likely that the RNase and proteins to require DsbC for proper folding of the isomerase activity of DsbC, although the of of the two redox active cysteines of DsbC in these proteins is not known A. J.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). than these three E. coli the protein native to the bacteria with than two cysteines for DsbC phosphatase, two consecutive disulfide bonds and was not the AppA homolog, Agp, with a to the role of nonconsecutive disulfide bonds in DsbC Agp and AppA show the of amino acid sequence, and are In Agp contains three disulfide bonds similar in the and as three of the disulfide bonds in AppA. However, Agp is the disulfide bond found in which is the nonconsecutive of Agp in cells for showed in activity of the protein with a Thus, we it likely that the nonconsecutive disulfide bond of AppA is the AppA Because Agp and AppA are similar in structure, it likely that between the two proteins a role in the in the we were that explanation for the between Agp and AppA in DsbC dependence is that the in of disulfide bonds in the and in the is However, it is known that proteins with three disulfide bonds be dependent on DsbC (11Rietsch A. Bessette P. Georgiou G. Beckwith J. J. Bacteriol. 1997; 179: 6602-6608Crossref PubMed Scopus (194) Google Scholar, J.C. Swartz J.R. Biochemistry. 1997; 36: 10067-10072Crossref PubMed Scopus (118) Google Scholar). Furthermore, activity has two nonconsecutive disulfide bonds and is dependent on DsbC (38Joly J.C. Swartz J.R. Biochemistry. 1997; 36: 10067-10072Crossref PubMed Scopus (118) Google Scholar). to this we a of of AppA in which a of cysteines in each of the disulfide bonds was to of these proteins one of disulfide bonds AppA enzymatic mutant each of which one of the consecutive disulfide dependence on DsbC. Thus, proteins with three disulfide bonds show dependence on DsbC. A mutant lacking the nonconsecutive disulfide bond of AppA was dependent on DsbC. The lacking a consecutive disulfide not to be but its AppA activity was low that the of this is not we were to Agp a protein that was to one that required DsbC for into active enzyme by two cysteines into the protein that could a disulfide bond to the nonconsecutive disulfide Because it is likely that this of Agp in in of the protein we it is the not in structure, that to the DsbC It is that proteins with disulfide bonds detected in E. cells to of proteins and membrane are other DsbC substrates to be that for an E. coli protein to be a substrate of DsbC it requires disulfide we the for It was for to to likely periplasmic membrane and cytoplasmic membrane proteins disulfide bonds. the proteins as a cysteine and or of we not whether the proteins form disulfide bonds. these that of periplasmic proteins the to be misoxidized by DsbA and require the isomerization of DsbC to be the disulfide AppA has to a nonconsecutive disulfide Although the that are with explanation for DsbC dependence of are for that the be in the of the DsbA catalyzes the formation of a C-terminal disulfide bond to the formation of the N-terminal disulfide bond P. C. E. M. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). disulfide bond formation is dependent on the folding of and co-workers P. C. E. M. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) that the N-terminal disulfide bonds of are not formed the proper folding of the is in of the Thus, disulfide bonds be formed the protein a structure, which not the C-terminal disulfide has proteins to require for folding pathway the formation of disulfide bonds between cysteines that are not found in the active protein J. Mol. Biol. 179: PubMed Scopus Google Scholar). the between cysteines that are to be in a consecutive disulfide bond and the of one or both of to other cysteines the that DsbA In this proteins be found that show consecutive disulfide bonds in but still DsbC of folding of the protein during the translocation cysteines that are not to be in the is the in vitro, folding of the protein in the of is necessary for the formation of native disulfide bonds Biochemistry. PubMed Scopus Google Scholar). In the of is and the protein is a disulfide bond although AppA activity DsbC is still a enzymatic activity of that found in a could be to one of is low isomerase activity in the be as the mutant the activity as the However, when is it AppA D. and J. The of of in AppA in be to low of is expressed the of DsbC Georgiou G. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google or low for AppA. of these be by or could be a in the or periplasm that misoxidized DsbA to form the correct bonds. The of formation of incorrect disulfide bonds by DsbA when a protein with a nonconsecutive disulfide bond not be of proteins of the of the DsbA has on The AppA activity that in the mutant not be to protein with correct disulfide bonds but to one or of AppA incorrect disulfide bonds that still enzymatic are not folded results activity of mutant proteins one or disulfide bond is with this We G. for and we the of the Beckwith
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